Dual T-cell depletion with individually tailored anti-thymocyte globulin and attenuated dose of post-transplant cyclophosphamide in haploidentical peripheral stem cell transplantation

This study aimed to assess the efficacy of dual T-cell suppression using individually tailored doses of antithymocyte globulin (ATG) and attenuated dose of post-transplant cyclophosphamide (PTCy) in haploidentical hematopoietic stem cell transplantation (haplo-HSCT). We conducted a retrospective analysis of 78 adults with acute leukemia or myelodysplastic syndrome who underwent haplo-HSCT using intravenous busulfan and fludarabine conditioning. Thirty-two patients received attenuated ATG/PTCy, while 46 patients received ATG (7.5 mg/kg) as GVHD prophylaxis. The 100-day cumulative incidence of grade III-IV (9.7% vs. 32.4%, P = 0.018) acute GVHD, as well as 2-year moderate-severe chronic GVHD (13.9% vs. 43.9%, P = 0.018) in the ATG/PTCy group were significantly lower than those in the ATG group. The 2-year overall survival was comparable between the two groups. However, 2-year GVHD-free, relapse-free survival in the ATG/PTCy group was significantly higher compared to that in the ATG group (38.9% vs. 21.7%, P = 0.021). Moreover, during post-engraftment period, the ATG/PTCy group exhibited lower incidences of life-threatening bacterial (12.5% vs. 37%, P = 0.033) and viral infection (0% vs. 17.4%, P = 0.035) than the ATG group. In conclusion, the combination of individually tailored ATG and low-dose PTCy appears to be a promising strategy in haplo-HSCT.


Participants and study design
This is a retrospective study of patients with acute leukemia and MDS who were over 18 years old and underwent haplo-HSCT following identical busulfan and fludarabine (BuFlu) conditioning between June 2017 and 2023 at Seoul National University Hospital, Seoul, South Korea.All patients received either ATG (ATG group) or a combination of ATG and PTCy (ATG/PTCy group) as T-cell depletion for GVHD prophylaxis.In the consideration of the choice between ATG and ATG/PTCy, some of the staff physicians implemented ATG/PTCy for research purposes, while the remainder adhered to the conventional institutional practice of applying ATG.Haploidentical donors were considered in cases where a matched donor was unavailable or when HSCT was urgently needed.In all patients, peripheral blood stem cells (PBSCs) were used as a graft source.

Transplantation procedures
For the ATG group, busulfan 3.2 mg/kg and fludarabine 40 mg/m 2 on days -6 through -3 were used as myeloablative conditioning (MAC), and busulfan 3.2 mg/kg on days -7 and -6 with fludarabine 30 mg/m 2 on days -7 through -2 were used as reduced-intensity conditioning (RIC).ATG (rabbit, thymoglobulin; Sanofi-Aventis, Korea) 2.5 mg/kg on days -3 through -1, for a total of 7.5 mg/kg was administrated as GVHD prophylaxis.For the ATG/PTCy group, the MAC regimen was the same as in ATG group, while for the RIC, busulfan 3.2 mg/ kg on days -6 and -5 with fludarabine 40 mg/m 2 on days -6 through -3 were employed.ATG was given from days -3 through -1 and was adjusted per absolute lymphocyte count (ALC) on days -3 based on our previous study 18 : for ALC > 1,000/μl patients, total of 4.0 mg/kg was used; for ALC 500-1,000/μl, total of 3.5 mg/kg; and for ALC < 500/μl, total of 3.0 mg/kg was used.PTCy was administered at doses of 50 mg/kg on day 3 and 30 mg/ kg on day 4 (Supplementary Fig. 1).In addition, calcineurin inhibitors were used to prevent GVHD.The choice of calcineurin inhibitors, either cyclosporine or tacrolimus, was left to the attending physician's preference.Methotrexate (15 mg/m 2 intravenous push on day 1 and 10 mg/m 2 on day 3 and day 6) was considered in ATG group, and mycophenolate mofetil (15 mg/kg three times a day from days 5 through 30) was administered in ATG/PTCy group.
All patients received uniform prophylactic regimens; ciprofloxacin for gut decontamination, trimethoprim/ sulfamethoxazole for Pneumocystis jirovecii, micafungin for fungus, and acyclovir for herpes simplex virus.The serum CMV polymerase chain reaction test was conducted twice weekly, and preemptive ganciclovir treatment was initiated in cases of CMV reactivation.

Definitions and outcomes
The diagnosis of acute leukemia and MDS was made according to the revised 2016 WHO classification.The modified European Group for Blood and Marrow Transplantation (mEBMT) risk score was used to assess risks of HSCT 19 .The 2016 MAGIC criteria were used to grade aGVHD 20 .Chronic GVHD (cGVHD) was classified as mild, moderate, or severe according to the 2014 National Institutes of Health consensus criteria 21 .Neutrophil engraftment was defined as absolute neutrophil count (ANC) > 500/μl for 3 consecutive measurements.Platelet engraftment was defined as 3 consecutive measurements of > 20,000/μl without transfusion.Non-relapse mortality (NRM) was defined as death without progression of underlying disease.Relapse-free survival (RFS) was defined as the time from stem cell infusion to relapse or death from any cause.Overall survival (OS) was defined as the time from stem cell infusion to death of any cause.GVHD-free, relapse-free survival (GRFS) was defined according to Ruggeri et al. (ie, being alive with no grade 3-4 aGVHD, severe cGVHD, or relapse at any time point after HSCT).The cause of death was evaluated as primary disease, graft failure, GVHD, infection, and other based on a previous report 22 .Any bacterial, viral, and fungal Infection data were collected retrospectively until the patient's death or last follow-up, using standardized definitions of severe infections after HSCT 23,24 .Infections requiring intensive care or vasopressor support were considered life-threatening infections.

Statistical analysis
Differences between groups were assessed using a Student's t-test or Wilcoxon rank-sum test for continuous variables, and chi-square test for categorical variables, as indicated.Median follow-up duration was calculated by the reverse Kaplan-Meier method.RFS, OS and GRFS were estimated by the Kaplan-Meier method and logrank test was used for univariate comparisons of survival.Cumulative incidence curves were used in competing risk setting to calculate the probability of GVHD, relapse incidence and NRM.Univariate and multivariate Cox proportional hazards regression models were used to analyze GVHD and survival outcomes with type of GVHD prophylaxis, age, diagnosis, mEBMT score, conditioning intensity, amount of infused CD34 + cell, and HSCT timing.Multivariate analysis was performed using stepwise backward selection, and adjusted hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated.All tests were 2-sided, and P values of < 0.05 were considered statistically significant.For all parts of statistical analyses, the statistical software 'R' version 4.3.1 (www.r-project.org) were used.

Ethics declarations
This study was conducted according to Declaration of Helsinki and was approved by the Institutional Review Board of Seoul National University Hospital (IRB no.H-2207-083-1339).The requirement for informed consent was waived by the Institutional Review Board of Seoul National University Hospital due to the retrospective nature of the study.

Patient and disease characteristics
A total of 78 patients were included in this study; 32 belonged to the ATG/PTCy group, while 46 were in the ATG group.Table 1 presents the baseline characteristics of the two groups.There were no differences in baseline characteristics, including disease status and infused CD34 cell dose, between the two groups.
Up to December 2023, all of the patients were followed for a median of 38 months (95% CI, 29.4-44); the median follow-up time for ATG/PTCy and ATG groups were 29.4 and 43.3 months, respectively.During followup, 37 patients (47.4%) relapsed and 47 (60.3%) died.There was no difference between the groups in terms of causes of death.
In pre-engraftment period (≤ day 30), there was no significant difference in the incidence of infection between the ATG/PTCy and ATG group (34.4% vs. 54.3%,P = 0.131) (Table 4).The 30-day cumulative incidences of severe bacterial, viral, and fungal infections were comparable between the groups (Supplementary Fig. 2).In the ATG/ PTCy group, a lower proportion of patients experienced two or more infectious episodes compared to the ATG group (3.1% vs. 21.7%,P = 0.046).

Discussion
In patients with acute leukemia and MDS undergoing haplo-HSCT using PBSC and BuFlu conditioning, this study demonstrated the efficacy of our novel GVHD prophylaxis protocol (combining low-dose PTCy and ATG adjusted for days -3 ALC levels) in reducing severe GVHD compared to the standard ATG-based regimen.Dual T-cell depletion with individually tailored ATG and attenuated dose of PTCy did not elevate relapse risk and improved GRFS.Additionally, ATG/PTCy resulted in fewer severe infections than ATG.
Due to PTCy's potential to enhance the effect of ATG in mitigating GVHD 17 , recent studies have explored various combinations of ATG and PTCy to improve GVHD prevention in haplo-HSCT (Supplementary Table 3).The cumulative incidence of aGVHD with ATG/PTCy in our study was comparable with other reports, indicating 11.5-34.6%for grade II-IV and 5-9% for grade III-IV aGVHD [25][26][27][28] .These studies consistently suggest that,  despite variations in dosing regimens, dual T-cell depletion with ATG/PTCy can be more effective in reducing severe aGVHD than ATG alone.Additionally, we found a significantly lower cumulative incidence of moderatesevere cGVHD for ATG/PTCy.As the mechanisms of GVHD prevention differ between ATG and PTCy 29,30 , the synergistic effect of the ATG/PTCy combination can be influenced by the dosage and timing of each.Studies with PTCy at a total dose of only 29 mg/kg 26 and ATG used post-transplant at a dose of 2.5 mg/kg 25 showed no difference in the incidence of moderate-severe cGVHD between the two groups.In contrast, a study using a total PTCy dose of 80 mg/kg (with a total of 7.5 mg/kg of pre-transplant ATG), similar to ours, demonstrated  www.nature.com/scientificreports/ a fewer extensive cGVHD in the ATG/PTCy group 27 .Furthermore, in a study using ATG 5 mg/kg and PTCy 50 mg/kg, the ATG/PTCy group had less moderate-severe cGVHD 31 , consistent with our findings.In our study, ATG/PTCy significantly reduced GVHD but did not impact the relapse risk.Instead, we observed a significantly higher GRFS in the ATG/PTCy group, consistent with previous studies 25,26 .Our result demonstrated that dual T-cell depletion with ATG/PTCy effectively reduces the risk of severe GVHD without increasing the relapse risk, resulting in a favorable GRFS outcome.Further mechanism studies on the dosing of ATG and PTCy are warranted to achieve the optimal synergistic effect of ATG/PTCy protocol.Excessive exposure to ATG may increase immunosuppressive toxicity, elevating risks of infections and relapse 32 .This risk could be heightened when used with PTCy, emphasizing the need for a strategy to judiciously reduce the dose of ATG 26 .Previous studies reported that weight-based dosing of ATG carries the risk of overdose, and the ALC level on the day of ATG administration could optimize ATG dosing 18,33 .Our ATG/PTCy protocol tailored the dose of ATG individually for each recipient based on the ALC level at day -3; i.e., if the ALC is low,  www.nature.com/scientificreports/ a small amount of ATG is administered.The combination of low-dose ATG and PTCy did not increase CMV infection compared to the standard dose of ATG, consistent with previous studies 25,31 .Additionally, we found a significant decrease in life-threatening bacterial and viral infection in the ATG/PTCy group, particularly in the post-engraftment period.As ATG is associated with a delay of the immune reconstitution, an attenuated dose of ATG might ameliorate post-engraftment viral infections 34 .Moreover, the ATG group exhibited more moderatesevere cGHVD, suggesting the potential for prolonged immunosuppressive therapy.These factors could have contributed to a higher incidence of bacterial infections in the ATG group.The reduced risk of life-threatening infections might have been more attributed to individually tailored ATG dosing than to the synergistic effect of ATG and PTCy.Further research is needed on personalized dosing in GVHD prophylaxis with ATG/PTCy for haplo-HSCT.This study had several limitations.First, this is a retrospective study.Thus, the two groups were not randomized and characteristics between the two groups were not fully comparable, although generally well-balanced.Although ATG/PTCy was identified as an independently favorable factor in the multivariate analyses, there is a possibility of confounding effects due to patient selection and differences between the groups, such as the use of additional immunosuppressants and timing of HSCT.Second, a relatively small sample size may limit the statistical power.Instead, we minimized the confounding factors; diseases included only MDS and acute leukemia, and all patients received an identical conditioning regimen and graft source, and infection prophylaxis.Third, we did not investigate data on immune reconstitution and ATG concentration.For the optimal dose and schedule of ATG/PTCy, further mechanism studies are warranted.
Despite these limitations, our study implemented a novel personalized dosing strategy by combining lowdose PTCy with a reduced dose of ATG tailored to the ALC level on the day of ATG infusion.We compared its outcomes with those of an ATG-based regimen in haplo-HSCT with the same graft source and conditioning regimen.Additionally, we conducted a comparative analysis of infection episodes, identifying differences between the two groups in terms of timing and etiology of infections.
In conclusion, our results demonstrated that dual T-cell depletion with an individually tailored ATG and attenuated dose of PTCy led to a low incidence of both severe aGVHD and cGVHD, improving GRFS compared to the standard-dose ATG regimen.In addition, ATG/PTCy showed a low incidence of severe infections, making it a feasible protocol.Reducing the PTCy dosage slightly and incorporating an individually tailored small amount of ATG appears to be a promising strategy in haplo-HSCT, emphasizing the need for further research to determine the optimal regimen. https://doi.org/10.1038/s41598-024-64361-5www.nature.com/scientificreports/

Figure 1 .
Figure 1.Graft-versus-host disease (GVHD) outcomes according to treatment groups.(A) Cumulative incidence of grade II-IV acute GVHD, (B) Cumulative incidence of grade III-IV acute GVHD, (C) Cumulative incidence of moderate-severe chronic GVHD.

Figure 2 .
Figure 2. Survival outcomes according to treatment groups.(A) Kaplan-Meier curves of overall survival, (B) Kaplan-Meier curves of relapse-free survival, (C) Kaplan-Meier curves of GVHD-free, relapse-free survival, (D) Cumulative incidence of relapse, (E) Cumulative incidence of non-relapse mortality.

Figure 3 .
Figure 3. Cumulative incidence (CI) of post-engraftment infection according to treatment groups.(A) CI of severe viral infection, (B) CI of life-threatening viral infection, (C) CI of life-threatening bacterial infection.